ABSTRACT Dental caries is the most common infectious disease in the world caused in large part by the Gram-positive bacterium Streptococcus mutans. Associated annual health care costs tens of billions of dollars, and rates of childhood caries in the U. S. are rising. There is a clear imperative to address this unmet health care need and identify new approaches to stem caries pathogenesis. Organisms that cause cavities form recalcitrant biofilms, generate acids from dietary sugars, and tolerate acid end products. While amyloid was first identified in the context of pathology, it does not always represent a protein mis-folding pathway. Functional amyloid is now recognized. Amyloid represents an evolutionarily conserved fibrillar, cross ?-sheet quaternary structure in which the ?-sheets laterally self-assemble to form fibers. Amyloid aggregates have common biophysical properties including uptake of amyloidophilic dyes, typical diameters when viewed by transmission electron microscopy, and birefringent properties when stained with Congo red and viewed under cross-polarized light. Several microorganisms are now known to produce functional amyloids that are integral to biofilm development. Our group was the first to identify Streptococcus mutans as an amyloid-forming organism. We have now extended that work to identify a total of three amyloid forming proteins in this bacterium. We have provided extensive tertiary and quaternary structural characterization of adhesin P1 and have identified its carboxy-terminal C123 truncation derivative as the amyloidogenic moiety. We have also identified S. mutans WapA and a previously uncharacterized protein Smu_63c as amyloid forming proteins. Like P1, WapA is a surface-localized sortase substrate whose truncation derivative, AgA, is amyloidogenic. Smu_63c is a secreted protein that serves as a negative regulator of genetic competence and biofilm cell density. All three of these proteins contribute to S. mutans biofilm development, which can be inhibited by small molecule inhibitors of amyloid fibrillization. Biofilm development by S. mutans mutants lacking genes encoding the amyloid forming proteins is significantly less susceptible to inhibition by compounds that target amyloids indicating the feasibility of amyloid inhibition as a therapeutic approach to preventing biofilm formation by this pathogen. In this renewal application we will utilize state of the art methods, including solution and solid state NMR, to identify and characterize the structural transitions underlying amyloid fibrillization by the amyloid forming proteins of S. mutans (Aim 1). This will enable us to follow their structural progression from monomer to amyloid within biofilms and will reveal mechanisms of action of inhibitory compounds. We will also develop and optimize methods to differentiate monomeric and fibrillar forms of the S. mutans amyloid forming proteins in vitro and within biofilms (Aim 2). Lastly we will apply these detection methods to assess and identify relevant environmental triggers that regulate protein monomer to amyloid conversion within S. mutans biofilms (Aim 3), a process that is not yet well understood in any system studied to date.